Identification of novel regulators of lymphocyte development and differentiation using CRISPR-Cas9 screens

T helper-2 (TH2) and type 2 innate lymphoid cells (ILC2s) play important roles in parasite expulsion, wound healing and allergic diseases through the expression of type 2 cytokines including IL-4, IL-5 and IL-13. Central to lymphocyte function are molecules required for differentiation and development. Several lineage-specifying transcription factors and other signalling molecules are known to be important for TH2 cell and ILC2 differentiation and function. However, whether additional molecules participate in these processes is unclear. In this thesis, by performing CRISPR-Cas9 screens on primary mouse lymphocytes, novel regulators of TH2 cell differentiation and ILC2 development were identified. Genome-wide screens for regulators of IL-13 production by TH2 cells corroborated the roles of known molecules and additionally identified novel players such as integrin αv, Acly, ADNP and ZIP7. Notably, small molecule inhibitors and neutralising antibodies confirmed a previously unappreciated, but critical role of αv, in combination with integrin β3, in TH2 cell differentiation. The generation of mice with a conditional deletion of αv in T cells corroborated this essential role in TH2 cell differentiation. To screen for novel regulators of ILC2 development, a new ILC culture system was optimised in which purified common lymphoid progenitors (CLPs) could be transduced with CRISPR- Cas9 gRNA libraries prior to differentiation into ILC1, ILC2 and ILC3 subsets in an in vitro co-culture system. Using IL-13 expression as a readout, Nfkb2, Mef2d, Zfp871 and ADNP were identified and validated as novel regulators of CLP to ILC2 development and differentiation. Furthermore, screens using CLP from transcription factor reporter mice (including Id2, T-bet, Gata3 and RORγt mice) identified additional potential novel regulators of the ILC developmental network. The application of functional CRISPR-Cas9 screens in primary immune cells has successfully identified new regulators of lymphocyte development and differentiation, some of which may be potential targets for therapeutic intervention in asthma and allergy.Croucher Cambridge International Scholarshi